The present invention relates to adjuvants which are intended to be attached to a molecule in order to improve its activity, in particular to increase the strength of the immune response. It also relates to complexes which contain such an adjuvant attached to an active molecule.
The active molecule can, in particular, be a protein, a peptide, a polysaccharide, an oligosaccharide or a DNA or RNA nucleic acid.
The development of vaccines which are perfectly defined and which lack pronounced side effects requires the use of immunizing antigens of low molecular weight such as peptides or oligosaccharides. These antigens of low molecular weight, and also certain antigens of higher molecular weight, such as bacterial wall polysaccharides, cannot, on their own, induce a lasting, powerful immune response. It is essential to link these antigens to carrier proteins by chemical means or by using genetic manipulation.
The carrier proteins which are currently employed are of two types:
tetanus and diphtheria toxoids: too frequent use of these carrier proteins risks jeopardizing a strong response to the hapten and risks the possibility of problems with immunotoxicity,
a membrane protein extract from Neisseria meningitidis (OMPC): consists of a membrane protein which is contaminated with lipids and LPS.
Patent EP-267 204 proposed using a support molecule which is intended to be coupled to an immunogen and which consists of an E. coli or salmonella membrane protein.
The Applicant has demonstrated that a protein which is extracted from the outer membrane of Klebsiella pneumoniae considerably improves the immune response to an antigen or a hapten when it is administered to a host at the same time as the latter. More particularly, an OmpA protein, the P40 protein of K. pneumoniae, can be used as an adjuvant in immunogenic complexes when it is attached to an immunogenic element.
The chemical conjugates which are derived by coupling peptides to the P40 give good results, and an assessment of the immune response shows antibody responses to these peptides which are greater than those which are observed when KLH or TT reference carrier proteins are used.
However, the peptide antigens are preferentially attached to the C-terminal part of the sequence, which is the most immunogenic part of the molecule (Puohiniemi, R et al., 1990, Infect Immu. 58, 1691-1696). This can present a serious problem in the case of fusion proteins which contain the complete P40 sequence. Therefore, use of a fragment of the sequence which supports the adjuvant activity would have a greater effect in minimizing the immunogenicity of the carrier protein and the risks associated with this immunogenicity.
For this reason, the present invention relates to an immunogenic complex of the type which comprises an immunogenic element which is attached to an adjuvant which increases the strength of the immune response, characterized in that the immunogenic element is an antigen or a hapten, and in that the adjuvant comprises at least a part of the P40 protein of Klebsiella pneumoniae or a protein which exhibits at least 80% homology, and preferably at least 90% homology, with the P40 protein.
In particular, the invention relates to an adjuvant which consists of a protein or a peptide having the P40 sequence which is substantially devoid of the immunogenic parts.
These P40 fragments according to the invention are, in particular:
the P40 sequence which lacks the immunogenic periplasmic C-terminal part,
a sequence which contains the third and the fourth extramembrane loops flanking an intramembrane sequence,
a sequence which contains one invariant extramembrane loop and the adjacent intramembrane sequence.
Those P40 sequences are defined as invariant extramembrane loops which are homologous with the sequences of the loops which are conserved between different enterobacterial species. The sequences of the extramembrane loops which are not conserved during the course of evolution are termed variable loops. The extramembrane loops are located in accordance with the Vogel and Jahnig model (1986, J. Mol. Biol., 190: 191-199), which relates to E. coli OmpA.
The choice of the fragments and, more particularly, the third sequence (amino acids 127 to 179) is based on the hypothesis according to which the invariant extramembrane loops (conserved between the OmpAs of the different enterobacteria) contain sequences which are recognized by immunocompetent cells, with these latter being able to have receptors which recognize these sequences.
The specific recognition of these sequences by antigen-presenting cells would make it possible to target antigens towards these cells and thus to induce an adjuvant effect.
For this reason, the invention also relates to an adjuvant product which consists of the sequence encompassed between amino acids 1 to 179 of the P40 protein of K. pneumoniae, or to a sequence which exhibits at least 80%, and preferably at least 90%, homology with the sequence which is encompassed between amino acids Nos. 1 and 179 of the sequence of the P40 protein of K. pneumoniae. 
The invention furthermore relates to an adjuvant which consists of the sequence which is encompassed between amino acids 108 to 179 of the P40 protein of K. pneumoniae, or to a sequence which exhibits at least 80% homology, and preferably at least 90% homology, with the sequence which is encompassed between amino acids nos. 108 and 179 of the P40 protein of K. pneumoniae. 
According to another aspect, the invention relates to an adjuvant which consists of the sequence which is encompassed between amino acids nos. 127 to 179 of the P40 protein of K. pneumoniae, or to a sequence which exhibits at least 80%, and preferably at least 90%, homology with the sequence which is encompassed between amino acids nos. 127 to 179 of the P40 protein of K. pneumoniae. 
The sequences ID No. 2, ID No. 4, ID No. 6 and ID No. 8 correspond to adjuvants according to the invention. This protein, and these peptide adjuvants, can, in particular, be prepared from membranes of bacteria of the species Klebsiella pneumoniae. The process then comprises the following steps:
a) precipitating the lipopolysaccharides by adding detergent and a salt of a divalent cation, and recovering the supernatant,
b) precipitating the proteins from the supernatant and resuspending the sediment,
c) chromatographing the suspension on an anion exchanger and recovering the fractions which contain the adjuvant product,
d) chromatographing on a cation exchanger and recovering the fraction which contains the adjuvant product,
e) concentrating the fraction obtained from step d) in order to recover an adjuvant product in the form of protein or peptide which is essentially free of liposaccharides.
Dialysis steps can advantageously be interposed between steps b) and c), and steps c) and d), respectively.
The invention also relates to immunogenic complexes which can be obtained using the different adjuvants.
The adjuvant can be attached to the immunogenic element by chemical coupling.
This covalent coupling of the peptide hapten to the adjuvant can be effected in a manner which is well known in the state of the art. Reagents which are appropriate for this purpose comprise, in particular, N-succinimide esters, carbodiimides, EEDQ (N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline) and the like.
The fragment of the P40 protein concerned, and the immunogenic element, can also be fused by means of genetic manipulation.
The fusion protein which is obtained between the fragment of the 40 protein and the immunogenic element can also be fused, by genetic manipulation, to a protein which is a receptor for a serum protein, in particular for human serum albumin.
The immunogenic element, an antigen or hapten, can, in particular, originate from viruses; those which may be mentioned are RSV (Respiratory Syncytial Virus) proteins or their fragments, for example protein G of RSV, or the hepatitis B antigen.
In the case of the RSV G protein, use may be made of the entire protein or of its fragments, where appropriate modified by point mutation or deletion.
The Applicant demonstrated that administration of a hapten coupled to a fragment of the P40 protein according to the invention resulted in a substantial increase in the immune response while limiting the risks of reactions against the adjuvant itself.
A process for increasing the immunogenicity of an antigen or of a hapten, characterized in that the said antigen or hapten is attached to an adjuvant which comprises all or part of the sequence of the P40 protein of Klebsiella pneumoniae, in the form of a complex as previously defined, is also part of the invention.
The invention also relates, therefore, to a vaccine, characterized in that it contains an immunogenic element attached to a fragment of the P40 protein which lacks a substantial part of the C-terminal sequence of the native P40 protein.
It also comprises pharmaceutical compositions which contain a complex which is formed between an adjuvant and an immunogenic element, as previously defined, and pharmaceutically acceptable excipients which are suited to administration of the complex by the parenteral and/or oral routes.
The invention also relates to the nucleotide sequences which encode the previously described peptides or proteins, and to the use of these sequences as a medicament. More particularly, such DNA sequences can be used in compositions which are intended for immunization by the intramuscular or intradermal route.
The examples which follow are intended to illustrate the invention without limiting its scope in any way.